Project Summary The faithful propagation of genetic material is essential to life. A central prerequisite to the copying of DNA is the dedicated assembly of replicative machineries at proper sites on the chromosome. This replication initiation event depends on multiple components, including: 1) initiators, ATPases that bind origins and recruit other proteins to the replication start site, 2) helicase-loaders, which deposit replicative helicases onto DNA, and 3) primases, enzymes that create short oligonucleotides for extension by DNA polymerases. A long-term objective of our research has been to understand the molecular structure/function relationships that govern the initiation of DNA replication. Many of the proteins responsible for initiation have been identified, and a preliminary framework for their action is in place. However, there remains a host of outstanding questions regarding how these factors act individually and cooperatively to construct a competent replication fork. Our prior efforts in this area have generated new mechanistic models for initiator action, helicase loading, and priming that we are now in an ideal position to test. Using a combination of structural, biochemical, and biophysical methods we aim to: 1) Define how ATP controls DNA binding and remodeling by prokaryotic initiators, 2) Determine how ATP regulates bacterial helicase-loader assembly and function, and 3) Establish how the bacterial primase synthesizes primers and is inhibited by disparate types of small-molecule agents, including the stringent response regulator, (p)ppGpp. Together, these efforts will impact a number of scientific fronts, from understanding how origins are engaged and restructured to promote replisome construction, to defining how chemical energy and small-molecule inhibitors control the function of dynamic macromolecular machines. Knowledge of these processes is necessary for understanding how cells ultimately avoid initiation errors linked to genomic instabilities, transformation, and neoplasia.